A cellular receptor is a protein molecule that is embedded in the plasma membrane or cytoplasm of a cell, and may bind to a mobile signaling molecule. The molecule that binds to a receptor is called a “ligand”, and may be a peptide (such as a neurotransmitter), a hormone, a pharmaceutical drug, or a toxin. When the ligand binds to the receptor, the receptor generally undergoes a conformational change, triggering a cellular response. However, some ligands act solely as antagonists, blocking receptors without inducing any response. Ligand-induced changes in receptors result in physiological changes that ultimately constitute the biological activity of the ligands. Although ligand binding is generally the trigger for receptor activation, some receptors are capable of producing a biological response in the absence of a bound ligand. These receptors are said to have “constitutive activity” or “baseline activity”.
Ligands may have different activities with respect to cellular receptor activation and/or inactivation. An agonistic ligand is able to activate the receptor and result in a biological response that is enhanced over the baseline activity of the unbound receptor. Many natural ligands are full agonists. A partially agonistic ligand does not activate the receptor thoroughly, causing responses that are smaller in magnitude compared to those of full agonists. An antagonistic ligand binds to the receptor but does not activate them. This results in receptor blockage, inhibiting the binding of other agonists. An inversely agonistic ligand reduces the activity of receptors by inhibiting their constitutive activity.
G protein-coupled receptors (GPCRs) are also known as seven-transmembrane domain receptors (7TM receptors), heptahelical receptors, serpentine receptor, and G protein-linked receptors (GPLRs). GPCRs comprise a large protein family of transmembrane receptors that sense molecules outside the cell, activating intracellular signal transduction pathways and, ultimately, cellular responses. G protein-coupled receptors are found only in eukaryotes, including yeast, plants, choanoflagellates, and animals. The ligands that bind and activate these receptors include small molecules, peptides, large proteins, pheromones, hormones, and neurotransmitters. G protein-coupled receptors are involved in many diseases, and are the target of approximately one-half of all modern medicinal drugs (Filmore, 2004, Mod. Drug Disc. 24-28).
GPCRs share a common structural motif, having seven sequences of between 22 to 24 hydrophobic amino acids that form α-helices, each of which spans the membrane. Each membrane-spanning segment is identified by number: transmembrane-1 (TM-1), transmembrane-2 (TM-2), transmembrane-3 (TM-3), transmembrane-4 (TM-4), transmembrane-5 (TM-5), transmembrane-6 (TM-6), and transmembrane-7 (TM-7). The transmembrane helices are joined by strands of amino acids between transmembrane-2 and transmembrane-3, transmembrane-4 and transmembrane-5, and transmembrane-6 and transmembrane-7 on the extracellular side of the cell membrane (these are referred to as “extracellular” regions EC-1, EC-2 and EC-3, respectively). The transmembrane helices are also joined by strands of amino acids between transmembrane-1 and transmembrane-2, transmembrane-3 and transmembrane-4, and transmembrane-5 and transmembrane-6 on the intracellular side of the cell membrane (these are referred to as “intracellular” regions IC-1, IC-2 and IC-3, respectively). The C-terminus of the receptor lies in the intracellular space, and the N-terminus of the receptor lies in the extracellular space.
There are several principal signal transduction pathways involving the G-protein coupled receptors, including the cAMP signal pathway and the phosphatidylinositol signal pathway (Gilman, 1987, Ann. Rev. Biochem. 56:615-649). When a ligand binds to the GPCR, a conformational change is triggered in the GPCR, which then acts as a guanine nucleotide exchange factor (GEF). The GPCR may then activate an associated G-protein by exchanging its bound GDP for a GTP. The G-protein α-subunit, with the bound GTP, may then dissociate from the β- and γ-subunits to further affect intracellular signaling proteins or target functional proteins directly depending on the α-subunit type (Gαs, Gαi, Gαq/11, Gα12/13).
Excluding odorant receptors, the human genome encodes roughly 350 GPCRs, which have hormones, growth factors, and other endogenous transmitters as ligands. Approximately 150 of the GPCRs found in the human genome have unknown functions. Those GPCRs for which the natural ligand is unknown are referred to as “orphan” receptors.
One of these orphan GPCRs is GPR35. This receptor was first cloned by O'Dowd and coworkers after a screen of a human genomic library (O'Dowd et al, 1998, Genomics 47:310-313). The GPR35 gene contains a single exon that encodes a predicted 309-amino acid protein (“GPR35a” hereafter—SEQ ID NO:1 for amino acid sequence), and is mapped to region 2q37.3 by fluorescence in situ hybridization. Subsequently, the GPR35 gene was identified in a 66-kb interval on chromosome 2 (Horikawa et al., 2000, Nature Genet. 26: 163-175). GPR35 expression was detected in all fetal and adult human tissues examined, with relatively higher levels in adult lung, small intestine, colon, and stomach. Recently, Okumura and coworkers (Okumura et al., 2004, Cancer Sci. 95:131-135) found that GPR35a and an alternatively spliced form of GPR35 (which contains 31 amino acids at the N-terminus of GPR35) are expressed in gastric cancers. The alternatively spliced form of GPR35 is designated GPR35b (SEQ ID NO:2 for amino acid sequence). The amino acid sequence and domains of GPR35a and GPR35b are shown in FIG. 1.
GPR35 is homologous to the P2Y purinergic receptor GPR23, for which the ligand is lysophosphatidic acid, and it shares a 30% identity with the putative cannabinoid receptor GPR55 (Guo et al., 2008, J. Pharmacol. Exp. Ther. 324 (1):342-351; Taniguchi et al., 2006, FEBS Lett. 580 (21): 5003-5008; Johns et al., 2007, Br. J. Pharmacol. 152 (5):825-31; Ryberg et al., 2007, Br. J. Pharmacol. 152 (7):1092-1101). Preliminary studies of GPR35 by mRNA expression showed that it is expressed predominantly in the immune and gastrointestinal systems with no detection observed in brain tissue (O'Dowd et al, 1998, Genomics 47:310-313). However, recent RT-PCR studies have confirmed the presence of GPR35 in dorsal root ganglion, the cerebellum and brain, and GPR35b was cloned from a human whole brain cDNA library (Guo et al., 2008, J. Pharmacol. Exp. Ther. 324 (1):342-351; Ohshiro et al., 2008, Biochem. Biophys. Res. Commun. 365 (2):344-348; Taniguchi et al., 2006, FEBS Lett. 580 (21):5003-5008).
Three compounds—kynurenic acid (2-carboxy-4-hydroxy-quinoline), zaprinast (5-(2-propoxyphenyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one), and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB)—were recently identified as GPR35 agonists (Taniguchi et al., 2006, FEBS Lett. 580 (21):5003-5008; Taniguchi et al., 2008, Pharmacology 82 (4):245-249; Wang et al., 2006, J. Biol. Chem. 281 (31):22021-22028). Kynurenic acid, a metabolite of tryptophan and inhibitor of the ionotropic glutamate receptor, was identified using an intracellular calcium assay that required the co-expression of a mixture of G proteins. Using an Aequorin bioluminescence reporter readout in CHO cells co-transfected with either human, mouse, or rat GPR35, EC50 values for receptor activation were determined as 40 μM, 11 μM, and 7 μM respectively (Wang et al., 2006, J. Biol. Chem. 281 (31):22021-22028). Results were confirmed by using a secondary GTPγs membrane binding assay in the absence and presence of pertussis toxin. These measured EC50 values are relatively high in comparison to values typically observed for the affinities of endogenous GPCR agonists, which routinely fall in the intermediate to low nanomolar range. As such, a “true” high affinity endogenous ligand that would deorphanize GPR35 remains to be discovered.
There is thus a need to identify potent GPR35 receptor agonists, which may be used to activate the receptor and shed light on the receptor function in different cells and tissues. There is also a need to further characterize the cellular processes controlled and regulated by the GPR35 receptor, and determine which diseases or conditions may be treated by an agonist of this receptor. The present invention addresses and meets these needs.